Research Article Optimization of Protease and Amylase Production by
Rhizopus oryzaeCultivated onBreadWasteUsing Solid-State
Fermentation
Olfa Benabda ,1,2 SanaM’hir ,1MariamKasmi,2,3WissemMnif ,4
andMoktarHamdi1
1Laboratoire d’Ecologie et de Technologie Microbienne (LETMi),
Institut National des Sciences Appliquees et de Technologie
(INSAT), 676 Centre Urbain Nord BP, University of Carthage, Tunis
Cedex 1080, Tunisia 2High School of Food Industry of Tunis (ESIAT),
University of Carthage, Tunis, Tunisia 3Laboratoire de Traitement
et Valorisation des Rejets Hydriques (LTVRH), Center of Water
Researches and Technologies (CERTE), University of Carthage,
Tourist Route Soliman BP, 273-8020 Nabeul, Tunisia 4University of
Manouba, ISBST, BVBGR-LR11ES31, Biotechpole Sidi 8abet, 2020
Ariana, Tunisia
Correspondence should be addressed to Olfa Benabda;
[email protected]
Received 11 June 2019; Accepted 17 September 2019; Published 21
October 2019
Academic Editor: Yiannis Kourkoutas
Copyright © 2019 Olfa Benabda et al. *is is an open access article
distributed under the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly cited.
*is research was carried for the coproduction of two industrial
enzymes: α-amylase and protease via SSF by Rhizopus oryzae on
humidified bread waste. Fermentation time, inoculum size, initial
moisture content, salt solutions, and the thickness of the
substrate were investigated one by one. Fungus culture was carried
out in sterile aluminum trays, and pH was adjusted to 5.5. *e main
results showed that the highest levels of enzyme production were
obtained at 120 h, 65% relative humidity, height media of 1 cm, 105
spore/g, and M-9 solution (g/L): NaH2PO4, 12.8; KH2PO4, 3; NaCl,
0.5; NH4Cl, 1; MgSO4 7H2O, 0.5; CaCl2 2H2O, 0.01. α-Amylase
(100U/g) and protease (2400U/g) produced by SSF from Rhizopus
oryzae (CH4) on BW as substrate are of great interest in industries
and could be valorized as enhancers of the bread making
process.
1. Introduction
Food waste occurs at different stages of production, pro- cessing,
retailing, and consumption since it is produced throughout the food
life cycle [1]. *e amount of food waste has increased in the last
25 years due to population and economic growth.*ese wastes could be
considered valuable by-products and could be a valuable resource
for the pro- duction of value-added molecules [2, 3]. Food waste
consists mainly of six types according to their source or origin:
vegetable trimmings and pulp, starch-based waste, fruit peels and
pulp, spent grains, meat and fish waste, and dairy waste [4,
5].
*e high carbohydrate content and the additional nu- trients of food
waste make them ideal media for microbial growth and enzyme
production as well as other high value- addedmolecules [6]. Bread
is among the major food waste in many countries around the world.
It is estimated that 1.2
million tons of bread are wasted every year, globally [7].
Actually, in Tunisia, more than 45 billion breads, subsidized by
millions of Tunisian dinars (34 million USD), are thrown, annually
in the street [8]. Wastage occurs at bakeries, retail outlets with
leftovers, and consumer households. So, there is a real need to
develop processes for reducing the associated problems with bread
waste formation. BW is a good fer- mentation feedstock also since
it is available year round and its availability is not associated
with the harvest season [9]. It is used for its high yielding
bioenergy substrates mainly for ethanol and biohydrogen production
[10]. *e work of Pleissner et al. [11] suggested hydrolysed BW for
bioplastic production by Halomonas boliviensis. According to the
findings of Zhang et al. [12], bakery waste was successfully used
as the nutrient-complete hydrolysates for the fer- mentative
production of succinic acid on Aspergillus awa- mori and A. oryzae.
*is substrate has also been used to produce amylase [13]. Many
studies have tried BW as raw
Hindawi Journal of Chemistry Volume 2019, Article ID 3738181, 9
pages https://doi.org/10.1155/2019/3738181
materials for enzyme production such as glucoamylase and protease
[9, 13–18] using Bacillus, 8ermomyces sp., As- pergillus, or
Monascus purpureus. In fact, BW is a successful material for enzyme
production for the following advan- tages: availability, cost, and
reduced contamination. Re- cently, Benabda et al. [19] reported
that they successfully used BW containing valuable components as
media for microbial growth.
Solid-state fermentation (SSF), an environmentally friendly
technique, which uses solid substrates at low moisture levels,
requires suitable solid substrate among agroindustrial materials
[20]. Various rawmaterials are used for SSF, for example, winery
and brewery waste, olive mill waste, sunflower cake, sugarcane
bagasse, fruit’s peels and pulps, cereal brans, and BW [21–25]. In
this process, mi- croorganisms grow and produce a wide variety of
products such as mushrooms [26], aroma [21, 27], microbial oil [28,
29], preservatives like fumaric acid [30, 31], enzymatic production
of wax esters [32], and enzymes [14, 27], thus reducing the cost of
production, as reported by these studies.
Among microorganisms, filamentous fungi are the best adapted for
SSF due to their mycelia mode of growth as well as their neutral
physiological capabilities [33]. In the present work, we used
Rhizopus oryzae as filamentous fungi for amylase and protease by
SSF on BW. *is fungus is able to efficiently grow on a wide variety
of substrates under op- erational conditions, thus furnishing
numerous bioproducts of interest, such as enzymes, organic acids,
aromatic com- pounds, and colorants. In previous study, M’hir et
al. [34] reported that Rhizopus oryzae produces proteases in
mixture with Enterococcus decreasing gluten concentration for
celiac persons. Proteases produced could be alternatives to
detoxify gluten used in food biotechnology [35]. Given these data,
we were interested to use Rhizopus oryzae in SSF for the pro-
duction of proteolytic enzymes. Moreover, the addition of amylases
in bakery products contributes to improve con- sumer acceptance
[36]. Fungal α-amylases were considered as safe additives [37].
*eir application is gaining an in- creasing interest since it
produces milder processing con- ditions, reduced the formation of
byproducts, and lowered the refining and recovery costs [38].
*erefore, BW can be used as a substrate for enzyme production as it
is carbo- hydrate-rich waste source. To the best of author’s knowl-
edge, this study is the first considering Rhizopus oryzae for
enzyme production on BW.
In this work, we attempt the coproduction of two im- portant
industrial enzymes: α-amylase and protease by Rhizopus oryzae
through SSF using BW as a substrate. *e objective of this study was
to optimize the parameters such as fermentation time, initial
moisture content, height of substrate, inoculum size, and salts’
addition. All of these affect concomitant hydrolytic enzyme
production in solid- state fermentation of BW. *ese parameters were
evaluated by step wise/single parameter optimization.
2. Materials and Methods
2.1. Microorganism. Rhizopus oryzae CH4, belonging to the Culture
Collection of the Laboratory of Ecology and
Microbial Technology, was used in this study. *e culture was
propagated on potato dextrose agar (PDA) (Oxoid) and stored at
4°C.
2.2. Inoculum Preparation. For inoculum preparation, Rhizopus
oryzae was grown on the PDA plate at 30°C for 7 days for complete
sporulation [34]. 9mL of sterile distilled water containing 9 g/L
NaCl was mixed and was added to the plate, and the spores were
scraped with an inoculating loop under aseptic conditions. *e spore
suspension was used as the inoculum for subsequent fermentation. *e
spore count was carried out using a Malassez counting chamber. Fer-
mentations were inoculated with spores at different levels.
2.3. Substrate Preparation. Commercial white bread waste was
purchased from a local bakery and was aged from 6 days (BW was
stored at room temperature in the bakery) (moisture: 15.2/100 g dry
basis, carbohydrate 49/100 g db, and starch 47.2/100 g db), ground
with a mortar, sieved to obtain a fine powder, and then filtered
with a colander (5mmmesh size). BWwas used at the same day of
collection to conduct following experiments.
2.4. Fungal Solid-State Fermentation and Condition Optimization. BW
powder was taken in rectangular trays of approximate dimension 286
mm× 120 mm× 80mm (about 250 g of BW in each one). Trays were closed
with aluminum foil, autoclaved at 121°C for 15min, cooled to room
tem- perature, and mixed thoroughly with sterilized distilled water
to reach appropriate moisture content. *e moistened powder was
spread evenly on the tray in layers, typically between 0.5 and 2.5
cm deep in tray with an open top and then inoculated with fungal
spore suspension of Rhizopus oryzae CH4 as described above and
recovered with alumi- num foil. 1mL of cryopreserved spores
solution of Rhizopus oryzae (1× 105 spores/mL) was inoculated in
the surface of bread waste.*en, themixture was homogenized with
sterile spatula.*e operation was carried out aseptically. Trays
were incubated at 30°C under static conditions. Figure 1
illustrates the bread waste SSF flow chart.
Factors like incubation period, moisture content, in- oculum size,
pH, substrate height, and salt effects influencing the secretion of
enzymes by R. oryzae under SSF were in- vestigated by adopting the
search technique-varying pa- rameters one at a time. Optimization
of the fermentation parameters for SSF was carried out to determine
the effect of each parameter on enzyme production. α-Amylase and
protease production was studied in SSF of BW, by altering various
physicochemical and cultural conditions with one at the time.
2.5. Enzyme Recovery and Assay. For isolation of enzymes produced
in SSF samples, 5 g of the fermented substrate was suspended in
50mL of distilled water at room temperature using a kitchen-type
blender to form a suspension of 100 g/L. After agitation in a
rotary shaker (170 rpm) for 1 hour at 30°C, the slurry was squeezed
through wet cheese cloth
2 Journal of Chemistry
followed by the centrifuging the whole content at 8000 g, 15min,
and 4°C to remove the insoluble matter, and the clear supernatant
obtained was used as the source of enzymes [39].
2.6. α-Amylase Assay. *e amylolytic activity was assayed using the
DNS method after crude extraction of fermented bread waste powder
[40]. α-Amylase activity was de- termined at 55°C for 30min by
measuring the release of reducing sugar from 1% soluble starch
(w/v) as substrate in 25mM sodium acetate buffer at pH 5 where
D-glucose was used as standard. One unit of alpha amylase activity
was defined as the amount of enzyme that released 1 μmol of
reducing sugars, expressed in glucose equivalent, per minute per g
of fermented substrate under the specified conditions.
2.7. Protease Assay. *e method mentioned by Han et al. [10] was
used to quantify protease activity with modification. Exactly, the
reactionmixture containing 0.3mL of 20% (w/v) azocasein (Sigma),
0.1mL of enzyme preparation, and 0.2mL of citrate buffer at pH 5.2
was incubated for 4 h at 45°C. *e reaction was stopped by adding
2mL of 10% TCA and stored at 4°C for 10min, and the test tubes were
centrifuged at 3500 g for 5min at 4°C. One unit of the enzyme
activity (U) was defined as an absorbance (440 nm) change of 0.01
after 4 h at 45°C [34, 41].
2.8. Statistical Analysis. *e data for all experiments have been
calculated from three replications, with the values presented as
the mean± SE (standard error).
3. Results and Discussion
3.1. Effect of Incubation Period. To investigate the effect of the
incubation time on the enzymatic activity of R. oryzae under SSF
fermentation, the temperature was 30°C, the inoculum size 105
spores/g, the initial moisture content 60%, the substrate height at
1 cm, and the pH at 5.5. In SSF fermentation, several factors are
to be optimized since they control the microbial growth and thus
the enzymatic pro- duction, namely, temperature, fermentation time,
moisture content, pH medium, inoculum size, carbon and nitrogen
sources, and salt concentrations.
Figure 2 shows the effect of fermentation time on the production of
both amylase and protease for 144 h. *e amounts of protease and
amylase production were of 1260U/g and 16.2U/g on 24 h,
respectively. *e highest activity was achieved within 120 h of
fermentation for both enzymes (2412U/g and 100U/g, respectively,
for protease and amylase). After this fermentation time, the
activity fell drastically. In general, long incubation period
results in an enzyme production decrease consequent to the
reduction of nutrients in the substrate as well as to the release
of proteases and the drop in the pH in the substrate [42, 43].
M’hir et al. [34] reported a protease activity value of 2.50U.
According to Hashemi et al. [44], SSF with Bacillus sp. recorded an
optimal amylase activity of about 3800U/L, whereas Ferreira et al.
[45] reported a value of 6500UI/L on SSF using R. oryzae on wheat
bran. Enzyme production by microor- ganisms is highly influenced by
media components and physical factors such as temperature, pH,
incubation time, and inoculum density [46]. Trying to produce
protease by A. flavus using SSF, Johnvesly et al. [47] reported
that the maximum protease production occurred during the 7th day of
incubation. Protease production from rice mill wastes by
Aspergillus niger in SSF increased with the incubation period [48],
in accordance with our results. In addition to in- cubation time,
protease production strongly depends on the extracellular pH which
affects several metabolic and enzy- matic processes and transports
various components across the cell membranes, thus influencing the
cell growth and metabolite production [49]. Moreover, Nafisa et al.
[50] reported a gradual decrease in protease units correlated to an
increasing incubation period. *is indicated the enzyme’s role as a
primary metabolite, being produced in the log phase of the growth
of the fungus for using nitrogenous source of BW [51].
Concerning α-amylase, the maximal productivity of α-amylase 100U/g
was achieved after 120 h (Figure 2(b)), which is two times higher
than the reported result of Han et al. [14] on the 5th day by A.
awamori using BW. Glu- coamylase production by A. awamori showed
similarity to our results, using pastry waste without nitrogen
supplements [52]. In fact, variation levels of glucoamylase
activity were influenced by a good balance of carbon, nitrogen, and
phosphor [52]. Furthermore, Melikoglu and Webb [25] mentioned that
near 95% of the starch has been consumed by 144 hours. *e highest
enzymatic activities (protease and glucoamylase) were measured
during this period.
Amylase assay
powder)
121°C 15 min
120h
Protease assay
SSF fermentation
Figure 1: Bread waste valorization flow chart in the present
study.
Journal of Chemistry 3
3.2. Initial Moisture Effect. Initial moisture content of bread
waste was adjusted to 45%, 50%, 55%, 60%, 65%, and 70% levels.
Incubation period was 120 h, and the temperature was 30°C with an
inoculum size of 105 spores at an inoculation height of 1 cm. *e pH
was fixed at 5.5. Our results showed that the protease activity
ranged from 1150 to 1550U/g, whereas that of α-amylase varied from
63.42 to 216.66U/g (Figure 3). A concomitant production for optimal
protease and amylase was, respectively, of 1550U/g and 216.66U
obtained with an initial moisture content of 65%. *e lowest ones
were associated with a common initial moisture content of
45%.
*ese results were consistent with levels mentioned by Haque et al.
[18], where optimum moisture content was about 65% for protease and
55–60% for glucoamylase ac- tivities. *ese authors reported a
maximum of 8U/g of glucoamylase and 116.6U/g for protease observed
with filamentous fungus Monascus cultivated in SSF on bakery waste.
*e protease activity showed in our study was more than tenfolds
higher than those reported by Haque et al. [18]. However, protease
values obtained by Haque et al. [18] are six times higher that
reported by Liang et al. [53], with the shrimp and crab shell
powder medium by Monascus. *e most important benefit of using bread
waste is its high nutrient content, making it useful without the
addition of supplements [19].
Higher initial moisture (180%) content has been re- ported by
Meligoklu et al. [16] with BW using A. awamori, where protease
production increased up to 71.6U/g, de- creasing after.
According to Sivaramakrishnan et al. [54], α-amylase production
increases with the increase of initial moisture content. A low
moisture level is associated with an early sporulation and a low
enzyme yield. *is may be explained by the nonavailability of
nutrients. A high moisture level
caused a decrease of particles porosity and a stickiness of
substrate, thus resulting in agglomeration and reduction of gas
volume and gaseous diffusion inducing as a consequence of low
oxygen transfer [16, 55].
Hence, it is important to provide the moisture content at an
optimum level. In SSF, water must be available in the
fermentationmedium for microbial growth and biochemical activity.
Indeed, water content influences the physical state of the
substrate, nutrient availability, diffusion of nutrients, and
oxygen-carbon dioxide exchange in a complex way [56].
3.3. Effect of the Inoculum Size. Inoculum amount is an important
biological factor, which determines biomass production in
fermentation [57, 58]. Maximum yield of enzyme production should be
result of a balance between the proliferating biomass and available
nutrients [59]. Figure 4 illustrates enzyme activity. BW was
inoculated with varied levels of spore suspension (104, 5.104, 105,
and 5.105 spores/g) with a moisture content of 65% and inoculation
height of 1 cm with pH 5.5. *e results showed maximal activities of
2400U/g db and 100.32U/g for both protease and amylase obtained
with an inoculum size of 105 spores/g. Tiann and Yuan [43] reported
that the inoculum level had no signif- icant influence on enzyme
productivity in SSF. However, Meligoklu et al. [16] reported
1.0million spores/g dry as optimum size. Demir and Tar [60],
determined that 107 spores/g is optimum for enzyme production in
wheat bran SSF by Aspergillus. Paranthaman et al. [48] have
mentioned that further increase in inoculum volume resulted in a
decrease in protease production.*is fact could be explained by high
amount of inoculum that caused overcrowding of spore thus and
decreasing enzyme pro- duction [59]. *e maximum protease production
was no- ticed at an inoculum size of 2×106 spores/mL [61].
0
500
1000
1500
2000
2500
3000
Pr ot
ea se
ac tiv
ity (U
A m
yl as
(b)
Figure 2: Effect of incubation period (h) on the production of
protease (a) and α-amylase (b) by R. oryzae CH4 at 30°C under
static conditions.
4 Journal of Chemistry
3.4. Effect of Salts. Experimental studies shown in Figure 5
presented that the optimal values of protease and amylase were
1493U/g and 90.06U/g, respectively, for salt solution S3.
Previously studies [62–64] have reported that NaCl (only present in
S3) enhanced protease production. *is salt plays an important role
in the stabilization and the protection of the enzyme from
undergoing denaturation [64]. Earlier studies reported that other
inorganic nitrogen sources in- crease enzyme production. In fact,
when supplemented with potassium nitrate, Aspergillus funiculosus
produced maxi- mum protease [65]. Furthermore, El Safey and Abdul
Raouf [66] and Karatas et al. [67] reported that ammonium sulfate
was the best nitrogen source for protease production. However,
Mukhtar and Haq [68] reported peptone as the best nitrogen supplier
for protease production by Rhizopus
oligosporus. Salt solution S3 with ammonium chloride in- creases
protease production by three times. *is result is in agreement with
the finding of Rathod and Pathak [69]. Presence of inorganic
nitrogenous supplements may im- prove nutritional quality and
consequently affect α-amylase synthesis. α-Amylase yield increased
with proper supple- mentation of C and N sources as they are
absolutely nec- essary for microbial growth [70]. Sindhu et al.
[71] have tried solid-state fermentation by Penicillium
janthinellum (NCIM 4960) using wheat bran. *ey reported that
α-amylase production was enhanced with ammonium sulfate, whereas
ammonium nitrate, ammonium chloride, sodium nitrate, potassium
nitrate, and sodium nitrite showed inhibitory effect on its
production. However, ammonium chloride induces production of
amylase [67]. *e inhibitory effects of
0
500
1000
1500
2000
2500
3000
Pr ot
ea se
ac tiv
ity (U
0
20
40
60
80
100
120
(b)
Figure 4: Effect of inoculum size on protease activity (a) and
amylase activity (b).
0
500
1000
1500
2000
2500
3000
Pr ot
ea se
ac tiv
ity (U
0
20
40
60
80
100
120
/g )
(b)
Figure 3: Effect of the initial moisture content (%) on the
production of protease (a) and α-amylase (b) by R. oryzae CH4 at
30°C for 120 h under static conditions.
Journal of Chemistry 5
some of the salts could be due to the pH changes in the
medium.
3.5. Effect of Substrate Layer 8ickness. *e thickness of the
substrate is an important factor and should be optimized for
controlling aerobic conditions for fungi. Protease and am- ylase
activities varied with substrate thickness that ranged
from 0.5 cm to 2.5 cm according to Figure 6. *e maximum protease
and amylase activities were recorded at the layer thickness of 1 cm
with respective values of 2400U/g and 100U/g. Similarly, results
were observed (11mm thickness) by Demir and Tari [72] with wheat
bran SSF by Aspergillus sojae for polygalacturonase production. In
fact, an increase of the layer thickness may reduce oxygen for
fungus in- ducing a poor growth [73]. However, the increase
of
0
500
1000
1500
2000
Pr ot
ea se
ac tiv
ity (U
(b)
Figure 5: Effect of salts on protease activity (a) and amylase
activity (b). S1: Czapek–Dox solution (g/l): NaNO3, 2.5; KH2PO4, 1;
MgSO4 7H2O, 0.5; KCl, 0.5. S2: M-15 solution (g/l): NH4NO3, 3;
KH2PO4, 1; MgSO4 7H2O, 1; FeSO4 5H2O, 0.01. S3: M-9 solution (g/l):
NaH2PO4, 12.8; KH2PO4, 3; NaCl, 0.5; NH4Cl, 1; MgSO4 7H2O, 0.5;
CaCl2 2H2O, 0.01. C: control: bread waste and sterilized distilled
water.
0
500
1000
1500
2000
2500
3000
Pr ot
ea se
ac tiv
ity (U
A m
yl as
(b)
Figure 6: Effect of substrate height on protease activity (a) and
amylase activity (b).
6 Journal of Chemistry
substrate thickness cannot promote fungal growth. On the contrary,
the lower thickness has been reported to be unable to support the
complete growth of fungus [74]. Dojnov et al. [75] reported that
high quantities of substrate enhance fungal growth. *e highest
α-amylase activity was detected on 32 g of triticale grains (medium
substrate height during SSF). According to these authors,
glucoamylase production was in correlation with fungal growth and
was the highest on 48 g of triticale grains (highest substrate
height).
4. Conclusion
BW is a good culture medium in SSF in terms of compo- sition and
safety. Enzymes production by Rhizopus oryzae such as α-amylase and
protease under optimized cultural conditions were as follows: 65%
moisture content, inoculum 105 spores/g, and incubation period of
120 h (5 days). Fur- ther studies should be continued to test
protease and α-amylase stability (thermal, pH, and detergent
stability) with the goal of performing their production in pilot
scale. Several other biochemical and biophysical parameters like
sensitivity towards ions, inhibitors, reaction kinetics, and
structural studies should be performed to validate their industrial
applications.
Data Availability
*e data used to support the findings of this study are available
from the corresponding author upon request.
Disclosure
*is research did not receive specific funding but was performed at
the Laboratory of Ecology and Microbial Technology (LETMi).
Conflicts of Interest
*e authors declare that there are no conflicts of interest
regarding the publication of this paper.
Acknowledgments
Special thanks are due to all the staff for their
cooperation.
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